Antimicrobial peptides from platelets (termed platelet microbicidal proteins [PMPs]) play a critical role in the innate immune system especially in the context of host defenses against endovascular infections. During endovascular colonization, the invading organism must contend with this limb of the innate immune system to persist and proliferate at such sites of infection. The mechanisms by which the organism can circumvent the effects of PMPs to survive within the endovascular space are not well understood. This is especially true of "adaptive responses" that enable PMP-susceptible organisms to survive exposures to these peptides. This proposal will employ three integrated specific aims to address our overall hypothesis: the cell membranes of endovascular pathogens such as Staphylococcus aureus are able to respond to and survive PMP exposures by a coordinated series of lipid biosynthetic adaptations, termed "homeoviscous adaptations". The three specific aims will be: 1) to fully characterize the homeoviscous cell membrane responses in vitro;2) to model these homeoviscous changes within customized liposomes ex vivo;and 3) to determine whether homeoviscous adaptations occur within relevant biomatrices and endovascular lesions in vivo. Aim 1 will utilize a series of in vitro techniques to examine the homeoviscous responses, including membrane fatty acid and phospholipid profiling, phospholipid assymetry and membrane fluidity. In addition, we will pair these analyses with a temporal assessment of expression by a series of prioritized membrane lipid biosynthetic genes. In Aim 2, we will employ large unilamellar vesicles whose membrane lipid contents will mirror those that emerge in vitro from Aim 1. These studies will define and quantify the impacts of homeoviscous adaptations on PMP:membrane interactions. Aim 3 will use the ex vivo biomatrix model and the rabbit model of endocarditis to explore the in vivo relevance of homeoviscous adaptations. The merging of in vitro-ex vivo-in vivo aspects of an important biologic event may well enable design of innovative strategies to prevent the organism from evolving these adaptations. This translational approach is within the mission of the NIH for their broad research roadmap. Lay Summary. The research in this proposal seeks to understand how bacteria can withstand the exposures to an important part of the host defense system (natural peptide antibiotics). This may enable design of new of strategies to kill such bacteria.